Abstract The broad, long term goal of the proposed research is to identify new therapeutic targets against HIV through the study of long non-coding RNAs (lncRNAs), a novel class of genes that due to their relatively recent discovery, have remained largely unstudied. Despite the availability of highly effective anti-retroviral therapies, eradication of HIV has remained impossible. This is largely due to the inability of existing therapies to target the latent reservoir of transcriptionaly silent HIV proviruses. While our understanding of HIV latency is far from complete, the existing data point to an important role for transcriptional and epigenetic regulatory programs in HIV latency. Interestingly, emerging data indicate that lncRNAs play critical roles in both transcriptional and epigentic regulation, pointing to the potential of this class of cellular RNAs as therapeutic targets against HIV. As a first step toward addressing this exciting possibility, we have taken a high throughput sequencing approach to define the extent of involvement of lncRNAs in HIV life cycle, targeting the clinically crucial steps of early infection, latency and reactivation. Our preliminary studies have revealed that all the above-listed stages of HIV life cycle are associated with differential expression of a large number of lncRNAs. Some of the differentially expressed lncRNAs originated from genomic loci that were positioned in close proximity of protein coding genes with key roles in HIV biogenesis. Intriguingly, in some cases, the architecture of the locus pointed to the possibility of a regulatory interaction between the lncRNA and the protein-coding RNA. Validation studies on two such loci proved the strong regulatory impact of the lncRNAs on their protein-coding neighbors. These exciting results led to the hypothesis that such local, cis regulatory networks may play an important functional role in HIV biogenesis and lead to identification of novel therapeutic targets. We propose to address this hypothesis under three specific aims. In Aim 1, we will complement our existing high throughput sequencing approach by including additional stages of HIV life cycle, including earlier time points of primary infection, and early and intermediate stages of latency and proviral reactivation. These studies will not only reveal the true extent of involvement of lncRNAs in HIV life cycle, but also they are likely to provide additional examples of potential cis-regulatory networks operating in HIV biogenesis. In Aim 2, we will test the validity of the potential cis-regulatory loci identified in Aim 1 using RT-PCR-based methods after forcefully increasing or decreasing the level of the lncRNAs in each locus using shRNA or transgene-mediated overexpression methods. Finally, in Aim 3, we will determine whether the validated cis-regulatory relationships impact HIV biogenesis. The most promising candidates with the strongest impacts on HIV biogenesis will be selected for additional studies to further evaluate their potential as anti-HIV therapeutic targets. Taken together, this project will define the extent of involvement of lncRNAs in HIV life cycle, and will identify cis regulatory networks that may impact the replication, latency or reactivation of HIV.